LOCUS       sequence-362297-        8574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of chimeric GA (N-eGFP-intron-C-mApple).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8574)
  AUTHORS   Xu B, Kucenas S, Zong H
  TITLE     zMADM (zebrafish mosaic analysis with double markers) for 
            single-cell gene knockout and dual-lineage tracing.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Mar 1;119(9). pii: 2122529119. doi: 
            10.1073/pnas.2122529119.
  PUBMED    35197298
REFERENCE   2  (bases 1 to 8574)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2022-03-1"; volume: "119(9). pii: 
            2122529119. doi"; pages: " 10.1073/pnas.2122529119"
COMMENT     Sequence Label: pTol2-eab2-GA
FEATURES             Location/Qualifiers
     source          1..8574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     369..386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    503..524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(539..544,545..562,563..569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    577..593
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     582..604
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     636..656
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        638..656
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     693..709
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     693..709
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(743..754,755..942)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    1381..1515
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1418..1437)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1472..1491
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1533..2240)
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAF
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNFE
                     DGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALKS
                     EIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1811..1830)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    2360..2393
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2394..2427
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2428..2461
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(2496..2529)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    2532..2563
                     /label=rox
                     /bound_moiety="Dre recombinase"
     primer_bind     2788..2809
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2851..2860
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(6358..6520,6521..6532)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     6881..6902
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7065..7169
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7170..7238,7239..8030)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7388..7407)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8201..8574,1..215)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
       61 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      301 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      361 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      541 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      601 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
      661 aaaagctgga gctccaccgc ggtggcggcc gctctagaac tagtggatct gctgggcttg
      721 ctgaaggtag ggggtcaaga accagaggtg taaagtactt gagtaatttt acttgattac
      781 tgtacttaag tattattttt ggggattttt actttacttg agtacaatta aaaatcaata
      841 cttttacttt tacttaatta cattttttta gaaaaaaaag tactttttac tccttacaat
      901 tttatttaca gtcaaaaagt acttattttt tggagatcac ttcattctat tttcccttgc
      961 tattaccaaa ccaattgaat tgcgctgatg cccagtttaa tttaaatgtt atttattctg
     1021 cctatgaaaa tcgttttcac attatatgaa attggtcaga catgttcatt ggtcctttgg
     1081 aagtgacgtc atgtcacatc tattaccaca atgcacagca ccttgacctg gaaattaggg
     1141 aaattataac agtcaatcag tggaagaaaa tggaggaagt atgtgattca tcagcagctg
     1201 cgagcagcac agtccaaaat cagccacagg atcaagagca cccgtggccg tatcttcgca
     1261 gatctgatct agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1321 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     1381 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1441 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1501 tatcatgtcg atatcaagta tacttacata tgttacttgt acagctcgtc catgccgccg
     1561 gtggagtggc ggccctcggc gcgttcgtac tgttccacga tggtgtagtc ctcgttgtgg
     1621 gacacgatgt ccaacttgat gtcgacgatg taggcgccgg gcagctgcac gggcttcttg
     1681 gccttgtagg tggtcttgac ctcggcggcg tagtggccgc cgtccttcag cttcagcctc
     1741 ttcttgatct cgctcttcag ggcgccgtcc tcggggtaca tccgctcctc ggaggcctcc
     1801 cagcccatgg tcttcttctg cattacgggg ccgtcggagg ggaagttggt gccgcgcagc
     1861 ttcaccttgt agatgaacac gccgtcctgc agggaggagt cctggttaac gtgaataatg
     1921 ccgccgtcct cgaagttcat cacgcgctcc cacctgaagc cctcggggaa ggacagcttg
     1981 aagtagtcgg ggatgtcggc tgggtgctta atgtagacct tggagccgta catgaactga
     2041 ggggacagga tgtcccaggc gaagggcagg gggccaccct tggtcacctt cagcttagcg
     2101 gtctgaaagg cctcgtaggg gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg
     2161 ttcacggagc cctccatgtg caccttgaag cgcatgaact ccttgatgat ggccatgtta
     2221 ttctcctcgc ccttgctcac ctaagggtgg gaaaatagac caataggcag agagagtcag
     2281 tgcctatcag aaacccaaga gtcttctctg tctccacatg cccagtttct attggtctcc
     2341 ttaaacctgt cttgtaacca taacttcgta taatgtatgc tatacgaagt tatataactt
     2401 cgtataaagt atcctatacg aagttatata acttcgtata atgtgtacta tacgaagtta
     2461 tgaagttcct ataccttttg aagaatagga acttcgaagt tcctatactt tctagagaat
     2521 aggaacttcg gtaactttaa ataattggca ttatttaaag ttaagatctt tgataccaac
     2581 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga
     2641 agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc
     2701 cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
     2761 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
     2821 ccccggtgaa cagctcctcg cccttgctca ccatggtggc ggatccactg taaaagaaag
     2881 ggaaactgtt agaattgcac tgctgccagg gagaggcgtt tcagcaagca tgtgaccatc
     2941 tggagtcaac ttcctgtttt gctctgcatt ctacagtctg accgctggtg ggcgctagtg
     3001 acagcgcgcg cctcagatat gccagacaaa ggaagtccct ctgcattctc tcattattac
     3061 cataaaaggc aatggtttga gccgctttgc ggctgccggc ggtgtggcac tcagccagag
     3121 tcgtagggct cagtaaaaag gaacagattt cgacattcat cacaccagga ccactcccta
     3181 aattttacac aaatgaagtt tcacatcctc ttccaccctc gcctgcgatc aattacacaa
     3241 ccgcatttca acgggtgtct gccgccgggt gttactgcgt cacagatctg cagtcagtcc
     3301 acggactcct cgtgagcgga gacttcacac taaagcattt ctggacggtc actctcagtc
     3361 ggttcagttc atagtctagc ctcaaaaatc accgagatcc tgaaaaactg acgggctctg
     3421 cttcgacagt cacagcggct gaagtgcagc cagagaactc cgcagccgtt attaaagtgg
     3481 accacacctc taaaattcaa aactctttca tagcggatac aatacttcac taacgttaat
     3541 cttcagcttc attaaagacg gagggggctg caggctacat ttaaatagaa cacataacaa
     3601 aaagaaaatt aaccaagttc aaactaaatg ccaatacccg ttactaataa caagatcttc
     3661 gatcttataa agcacgaatg ggccgatgtt agattgatgg ggtttacgtt acatcagatg
     3721 agagctggtt agccggctag ctaaagtagg tcacaccctg ctgcagcact gcctgcacaa
     3781 agaacttccc atgccggcta tccatcaagt gtgcaacatg ctcaccagca ccctcaaaac
     3841 tgtagttaaa aatcacattt atggctgttg aagagagatt agccagaaca caactttata
     3901 ttaagactta acacccattt cacaccggcc cattcaatgt tcaatgtcgt gcctacgact
     3961 ggtcacgtta aattaccgac attctcacct gccacatcca ctaacgctac aaaaaaagtt
     4021 aagatacagc caatatttat taaatgtaac agatattatc aaaagaacca ccatttataa
     4081 atcaaaccta gtagataact acaaagagac cgttatgaac tgtctaaaga cttcatcaaa
     4141 tcatatttat ccatttacac attttactta acattaatag atagaaacta taattaaacg
     4201 cttaaaaact gcaaaaccaa ctcaccttgg gtttgggggg ttgtcgactt aagagaatgg
     4261 gttacaagca gatgatattc cgcactagct gcgtgtggag gagctcaaag tgaggctgag
     4321 acgctgggcg cgctctttta tatgacggca catacccggc tctagccata aaaggtaaac
     4381 tttcggagct ctgtgctctg attggtccag cgtcacacac ccggctgtcg cgtgcacatt
     4441 gctacacttg cttcttgcaa cttgagagtg aaagaagggg aagcttaact ggttaaagaa
     4501 caaggccttg gcgatttcct ataaaactgg aatttttatt tgcaaatttt aacacagaat
     4561 ttttcgtatt tttctgcgac aacgccaaga ctcaattcaa cgtttcaaaa agtctgtgca
     4621 gagttacaag tggcagaatg cgtcataatg atggatgcct ccatgatcct gtgttgtgtt
     4681 ttcaatctaa cacaacagca gctctgtagg tcatttacct cttgtgttca cgaaataccg
     4741 taatacaaac aatgggggat ctaatgagta cttttccatg tataaaatgg acattttttt
     4801 tatccaaatt aggatgacac tacttgtagt atgttgtagg agcaactagc tggtcttagc
     4861 tcagtagtaa tgcactttaa gaaagtgctt taataacttt gaatgaatgg gacctgcatt
     4921 ccatgccaca ttgcttcact atttttgtgt gtacaagtta cacaatgatg tataaagtga
     4981 gtgatagcag ttacattttt gcaagtgggc ctacctcagg tttaatgttc attagaaatg
     5041 ttatatgtca agtcttggtc ttaaatagtt aaattttctg ttcataattt catgtcgtgt
     5101 ctatgcagac ataacatttc tttttaaaga ggtttataag gataaatcct aagaacataa
     5161 ttctcattaa aagtagaaaa aaaaacactg aaagtgttgg tgatcagcta caataaataa
     5221 tattttgcac acatttcaga tgttaccaca ctgaataaca gataaagctg tgcttgagca
     5281 gcagtgatca tgataaacta catgtgactt tttactgtgt aacttgggag taatgcacat
     5341 ggccttcagt gggagttata aacaaaaaca gctgattgtg ttaaaagtgc ttgctgatac
     5401 caaggacaat gaggtgatgt gacttttatt ttgttttcgg caaattccaa gtattttgac
     5461 aagacttttt acaattctga gcattttgcc tctaaaatat ggaagtttct cagagtaaac
     5521 gttgagtata ggtaatgcct aggccctgac tgtgcgcaca gaaatctgta gatttcccca
     5581 gatctttagc ccatcattga gtttatttat tgacttgtgt atttggattt gctctttagt
     5641 gtttggactc tcagcagtaa aaattaaccc acactgaact aaactgaact gaacttaaac
     5701 tcttaaaaat ggactgacac agtttcaatt tactatacta atcttctaag ttaagctgct
     5761 ttgacacaat ctacattgta aaagcactct agcttgcatg cctgagaatt tcagaatgta
     5821 atgatacctt tgttagataa ggctgtttac atctgatagt ggaccttaag ccgacactta
     5881 aatgataaaa acggcaaaga attgcaagtt tgatttgcat tggaaagggt cgctggcttt
     5941 tgtgttacac gccccttatt tgtgcttgat tagatgatcc ccctgctcga gggcccatct
     6001 ggcctgtgtt tcagacacca gggagtctct gctcacgttt cctgctattt gcagcctctc
     6061 tatcaagact aatacacctc ttcccgcatc ggctgcctgt gagaggcttt tcagcactgc
     6121 aggattgctt ttcagcccca aaagagctag gcttgacact aacaattttg agaatcagct
     6181 tctactgaag ttaaatctga ggttttacaa ctttgagtag cgtgtactgg cattagattg
     6241 tctgtcttat agtttgataa ttaaatacaa acagttctaa agcaggataa aaccttgtat
     6301 gcatttcatt taatgttttt tgagattaaa agcttaaaca agaatctcta gttttctttc
     6361 ttgcttttac ttttacttcc ttaatactca agtacaattt taatggagta cttttttact
     6421 tttactcaag taagattcta gccagatact tttactttta attgagtaaa attttcccta
     6481 agtacttgta ctttcacttg agtaaaattt ttgagtactt tttacacctc tgtcaagaac
     6541 tcctggacaa acctctgacc tgtgtggaac agagtggata tgggtgtctg aacagatatt
     6601 cacgtctttt gcagatcaga gggcatttct ggtgttgtgc aatgacctgg gtccaacagg
     6661 taggcgtact cgtatccaag agcattgttg gacaggaggt agtctccatt cctataaaga
     6721 ggaaggaatg gcaccatgta gtagccgtca ttgtggccaa ttggtgcatt ggtacgtggg
     6781 tagatggacc ggggaggctg atgagttctt agccagcgtg ggccatcgcc ctgatagacg
     6841 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     6901 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     6961 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     7021 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     7081 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     7141 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     7201 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     7261 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     7321 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     7381 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     7441 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     7501 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     7561 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     7621 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7681 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     7741 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     7801 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7861 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7921 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7981 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     8041 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     8101 aggtgaagat cctttttgat aatctcatga ccaaaatgcc ttaacgtgag ttttcgttcc
     8161 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     8221 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     8281 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     8341 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     8401 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     8461 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     8521 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctga
//
